REVIEW ARTICLE

https://doi.org/10.4110/in.2017.17.2.89 pISSN 1598-2629 · eISSN 2092-6685

Mesenchymal Stromal Cells and Toll-Like Priming: A Critical Review

Mehdi Najar1#, Mohammad Krayem2#*, Nathalie Meuleman1, Dominique Bron1 and Laurence Lagneaux1 1Laboratory of Clinical Cell Therapy, Institut Jules Bordet, Université Libre de Bruxelles (ULB), Campus Erasme, 2Laboratory of Oncology and Experimental Surgery, Institut Jules Bordet, Université Libre de Bruxelles, Brussels 1000, Belgium

Mesenchymal Stromal Cells (MSCs) are potential cellular candidates for several immunotherapy purposes. Their multilineage potential and immunomodulatory properties make them interesting tools for the treatment of various immunological diseases. However, depending on the local microenvironment, diverse biological functions of MSCs can be modulated. Indeed, during infections such as obtained following TLR-agonist engagement (called as TLR priming), the phenotype, multilineage potential, hematopoietic support and immunomodulatory capacity of MSCs can present critical changes, which could further affect their therapeutic potential. Thus, for appropriate clinical application of MSCs, it is important to well know and understand these effects in particular during infectious episodes and to find the suitable experimental settings to study that. Pre-stimulation of MSCs with a specific TLR ligand may serve as an effective priming step to modulate one of its function to achieve a desired therapeutic issue. [Immune Network 2017;17(2):89-102] Keywords: MSCs, TLR, Priming, Phenotype, Multilineage potential, Hematopoietic support, Immunomodulation

Mesenchymal stromal cells biological properties. From osteogenic stem cell or a bone marrow (BM) stromal cell (4), to Medicinal Signaling Mesenchymal stromal cells (MSCs) represent nowadays Cells as suggested by Caplan and Correa (5), the termino­ an important immunotherapeutic cell population with logy of MSCs varied across the time. A position statement several possible application for the treatment of immu­ released by the International Society for Cell Therapy nological-based diseases (1). MSCs are known as multi­ (ISCT) proposed to clarify the nomenclature of MSCs. potent, non-hematopoietic cells that can be found in This statement suggested that the fibroblast-like plastic- almost all tissues (2). Despite some common characteris­ adherent cells, regardless of the tissue from which they tics, source-dependent differences have recently emerged are isolated, should be termed multipotent mesenchymal and lead to different clinical applications of MSCs (3). stromal cells and thus keeping the acronym MSCs (6). Several reports have shown that differences in the Moreover, to better characterize MSCs and to standardize and transcriptomic profiles, as well as in the secretome the research in the field, the ISCT proposed a minimal and miRNome of MSCs may reflect differences in their set of standard criteria to define human MSCs (7). First,

Received on January 5, 2017. Revised on February 24, 2017. Accepted on February 25, 2017. This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons. org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. *Corresponding Author. Mohammad Krayem, Laboratory of Oncology and experimental surgery (LOCE), Institut J. Bordet, Université Libre de Bruxelles, Rue Héger-Bordet 1, 1000 Brussels, Belgium. Tel: 32-25413323; Fax: 32-25413349; E-mail: [email protected] #These authors contributed equally to this work.

Abbreviations: MSCs, Mesenchymal Stromal Cells; BM, bone marrow; HSCs, Hematopoietic Stem Cells; AT, adipose tissue; PGN, prostaglandin

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during culture MSCs must be plastic-adherent, present therapeutic strategy for immune-mediated diseases. a fibroblast like shape and able to give rise to CFU-F Mechanistically, immunomodulation occurs by different (colony forming unit-fibroblasts). Second, based on a pathways but two important sides have to be taken flow cytometry analysis of their immunophenotype, into account: the regulatory network of factors and the MSCs must be positive (>95%) for CD105, CD73 and gathering of regulatory immune cells. These pathways CD90, and negative (<5%) for CD45, CD34, CD14 (or compete to establish a tolerogenic state conducive for CD11b), CD79alpha (or CD19) and HLA-DR surface immunomodulation (16,17). molecules. Third, MSCs must differentiate in vitro to MSCs are also defined as environmentally responsive osteoblasts, adipocytes and chondroblasts under specific therapeutics as they are capable of responding to local culture conditions (8). Regarding their therapeutic environmental stimuli with a myriad of beneficial potential, several properties were associated with MSCs interventions (18). Indeed, MSCs were reported to be highlighting thus the importance of their use for different particularly sensitive to different environmental signals therapeutic purposes. Today, it is widely accepted that (19). Infection are known to be major events triggering MSCs are actively involved in the hematopoiesis support graft-versus-host disease (GVHD) after allogeneic stem (9,10). MSC are part of the highly specialized “bone cell transplantation (20). Mimicking infection through marrow microenvironment” and are critical for forming Toll-Like Receptor (TLR) activation has been shown to the niche that maintains Hematopoietic Stem Cells modulate the functions and responses of MSCs (21,22). (HSCs). MSCs actively participate in the regulation of In the following review, we discuss the importance to HSC survival, quiescence and, upon specific triggers, well study and understand the impact of infections via differentiation into mature cells (11) suggesting their TLR activation on the biology of MSCs particularly when role in the enhancement of hematopoietic engraftment therapeutic applications have to be proposed. Several during use in HSC transplantation (12). Moreover, it is MSC biological functions such as phenotype, multilineage known that under specific differentiating factors, MSCs potential, hematopoietic support and immunomodulatory could differentiate not only into tissues of mesodermal capacity have been observed to be drastically affected by origin, but also in other tissue lineage cells (13,14). specific TLR-agonist engagement (Figs. 1 and 2). A such multilineage potential is an MSC's hallmark allowing their use in regenerative medicine for different repair therapy indications (15). Finally, along with Sensitivity to infection their non-immunogenic state as indicated by the lack of HLA-DR expression, MSCs have the ability to pre­ In general, pathogenic infectious agents are detected sent a potent immunomodulatory potential allowing and destroyed rapidly by the defense mechanisms of to regulate both adaptive and innate immunity. This innate immunity providing thus the first barrier against unique feature leads to investigate MSC as a new cellular pathogens. Host-pathogen interactions are generally

Infections

TLR triggering

poly(I:C) Endosome SSRNA TLR7 TLR3 SSRNA TLR8 MSCs CpG-DNA TLR9

Fig. 1 Figure 1. Activation of TLRs on MSCs.

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initiated when host recognizes conserved molecular intracellular compartments (endoplasmic reticulum, structures that are essential for the life-cycle of the lysosomes, and endosomes) recognize viral nucleic pathogen and which are known as a pathogen-associated acids. molecular patterns (PAMPs) (23). PAMPs are sensed by TLRs are functional multimers and most of them are the host’s germline encoded pattern recognition receptors homomeric. However, TLR2, which strictly associates (PRRs), expressed by various immune cells such as with TLR1 or TLR6 (27) is considered heteromeric. dendritic cells (DC), macrophages or neutrophils (24- TLR4 is known to sense (LPS) which 26). When PAMPs are recognized by PRRs, an immune is an important component of cell walls of gram-negative response is triggered in the host through activation of bacteria. To be able to bind LPS and to initiate signal a complex signaling pathways which facilitates the transduction, TLR4 forms a complex with accessory eradication of pathogens (25). To date, several classes molecules such as myeloid differentiation-2 (MD2 of PRRs are characterized, and among them Toll-Like also known as Lymphocyte antigen 96), LPS-binding Receptors are the most widely studied. protein subunit (LBP) and CD14 (28). TLR2 senses peptidoglycan, and as a heterodimer with TLR1 (TLR2/ TLR1) or TLR6 (TLR2/TLR6) recognizes triacylated Toll-Like Receptors and diacylated lipopeptides respectively (29,30). TLR3 senses double-stranded RNA (dsRNA), TLR5 senses Nowadays, TLRs are considered as the primary sensors flagellin protein (31), TLR7 and TLR8 sense RNA and of pathogen presence and are involved in the immune TLR9 senses unmethylated CpG DNA fragments (26,32). response during infections. TLRs are type I trans- Despite extensive research on the TLRs, human TLR10 membrane glycoproteins with extracellular domain rich in has remained an orphan receptor without a known agonist leucine repeats that is responsible for PAMPs recognition. or function (33). The trans-membrane domains, and cytoplasmic Toll- Although TLRs are primarily thought to have evolved Interleukin 1 Receptor (TIR) domains are required for as sensors of exogenous stimuli, the recognition of endo­ downstream signaling. Until now, 10 functional human genous ligands is now considered to have an important TLR are described (24) depending on their cellular role in regulating the inflammation. These ligands are localization and the nature of PAMP ligands that they called “danger signals” as TLRs can sense them in the sense. setting of injury or non-infectious threat (34). Importantly, TLRs are divided into two sub-groups: TLR activation has been implicated in the pathology 1) ‌TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10 ex­ of various inflammatory diseases including rheumatoid pressed at the cell surface recognize microbial mem­ arthritis or inflammatory bowel disease (IBD), since they brane components: lipids, lipoproteins and . can either initiate or perpetuate the chronic inflammation 2) ‌TLR3, TLR7, TLR8 and TLR9 expressed only in due to the continuous exposure to TLR ligands (35- Endosome TLR7 TLR3 TLR8 MSCs TLR9

Phenotype Multilineage Hematopoietic Immunomodulatory potential support potential Figure 2. The MSC response following to TLR priming.

Fig. 2

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37). However, Fuenzalida et al. (38) has recently shown modulate the pattern and function of TLRs expressed that short in vitro TLR3 pre-conditioning with poly(I:C) by MSCs (53,54). BM-MSCs and adipose tissue (AT- enhances the therapeutic efficacy of UCMSCs (umbilical MSCs) shared the same TLR pattern featured with the cord matrix stem cells), which is a major breakthrough transcription of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6 for developing improved treatments for patients with and TLR9 and the absence of mRNA for TLR7, TLR8 inflammatory bowel disease. and TLR10. Of special interest, MSCs isolated from the umbilical cord matrix, called Wharton’s Jelly (WJ- MSCs) do not expressed TLR4 and expressed a non- TLR signaling functional TLR3. Cord blood derived MSCs (CB-MSCs), expressed low levels of TLR1,3,5,9 and high levels of TLR signaling is mediated by recruitment of different TLR4 and TLR6. Protein studies revealed that both TLR4 Toll-Interleukin 1 (IL-1) Receptor (TIR) domain-con­ and TLR5 were functional (55). Expression of TLR1- taining adaptor molecules. Currently, there are four 10 was confirmed in human amnion mesenchymal cytosolic adaptor proteins described: MyD88 (myeloid cells (AMC) but with different levels (56). Interestingly, differentiation primary response 88), TIRAP (TIR TLR6 and TLR9 were expressed at the highest levels domain-containing adapter protein), (TIR-domain- while the expression of the resting isoforms was very containing adapter-inducing interferon-b), and TRAM low. The relatively low expression and function of some (TRIF-related adaptor molecule) (24,31). Recruitment TLRs may be characteristic for MSCs originating from of these adaptor molecules leads to the activation of the early developmental stages. Dental MSCs were various transcription factors such as NFkB, IRF3/7, and showed to express mRNA encoding for TLR3 and TLR4 MAP kinases to induce the production of inflammatory (57). Moreover, hypoxia significantly increased mRNA cytokines and type I interferons (39). of TLR1, 2, 5, 9, and 10 (58) whilst infection of MSCs Signaling pathways activated by TLRs can be broadly with baculoviral vectors upregulated and activated TLR3 classified as MyD88-dependent and -independent signaling pathway (59). Recent study has shown that the pathways. TLR4 uses both signaling pathways whereas antibacterial effect of MSCs against Escherichia coli was TLR3 use only MyD88-independent one to activate IRF3 mediated by secretion of b- defensin 2 via TLR4 signaling and further to induce type I interferons transcription. All (60). Lastly, gingival margin-derived stem/progenitor cells the other TLR use MyD88-dependent signaling to activate (G-MSCs) showed a distinctive TLR expression profile. NFkB to induce transcription of pro-inflammatory Constitutively, G-MSCs expressed TLRs 1, 2, 3, 4, 5, 6, 7, cytokines (40). and 10. Inflammation significantly up-regulated TLRs 1, 2, 4, 5, 7 and 10 and diminished TLR 6 expression (61). Collectively, an inflammatory environment may modulate MSC and TLR expression the pattern and function of TLRs expressed by MSCs depending on their tissue source origin. TLRs are widely expressed by the main cells of the innate and adaptive immune system (41-43). TLR expression is not just a feature of immune cells as other cell types MSC phenotype and TLR-priming such as fibroblasts, epithelial, endothelial and MSCs may express several TLRs and thus contribute to the protection Within the immune system, TLR activation can modulate against infection (44,45). The first evidence that MSC the expression of Human leucocytes antigens (HLA) as express functional TLRs comes from the studies done well as several costimulatory molecules thus impairing in mice (46). Pevsner-Fisher et al. showed that mouse the immunological status and function of immune MSCs express functional TLR molecules 1 to 8, but not cells. Therefore, it is of great relevance regarding their TLR-9. In contrast, human MSCs expressed mRNA of use in allogeneic cell-based therapies to determine different TLRs. While the data concerning the expression whether MSC exposure to TLR ligands may induce of TLR1, TLR2, TLR3, TLR4, TLR5 and TLR6 are the expression of HLA-I, HLA-II, and costimulatory quite consistent, the expression of TLR7, TLR8, TLR9 molecules (CD40, CD80, CD86). Even in the presence and TLR10 is quite controversial and it seems that is of IFN-g (a well-known immunological inducer), MSC-origin dependent (21,47-52). As demonstrated activation of TLR4, TLR3 and TLR2 with LPS, Poly by Raicevic et al., several environmental conditions I:C, and PGN respectively had no significant effects on

92 IMMUNE NETWORK Vol. 17, No. 2: 89-102, April, 2017 TLR-priming of MSCs the immunogenic properties of MSCs from BM and AT receptors expressed on myeloid cells receptors (TREM-2) (47,49,62-65). Indeed, except induction of HLA-I by is constitutively expressed in BM-MSCs and that TREM- Poly I : C, none of the other ligands were able to alter 2 knockdown resulted in downregulation of several TLR the expression of HLA-II, CD80, and CD86. However, expression and inhibited osteogenic, chondrogenic, and another study demonstrated adverse effects of TLR3 adipogenic differentiation (72). Although the signaling and TLR4 activation on the immunological phenotype mechanisms remain unclear, these results strongly suggest of umbilical cord derived mesenchymal stem cells (UC- that TREM-2 activation is actively involved in the regula­ MSCs). They found that the expression of co-stimulatory tion of MSC multipotential potential. PGN (TLR2) and proteins CD80 and CD86 were unaffected following LPS (TLR4)-induced osteogenic differentiation has been treatment with the TLR3 agonist (Poly I : C). In the other demonstrated in stromal cells derived from human adipose side, only TLR4 ligand (LPS) increased the expression of tissue (hADSCs) following activation of NF-kB and CD86. Interestingly, the expression of CD74 (HLA class subsequent up-regulation of PDZ-binding motif (TAZ) II histocompatibility antigen gamma chain also known as expression (58). Lombardo and colleagues (47) showed HLA-DR antigens-associated invariant chain) and CD105 that TLR3 (Poly I:C) and TLR4 (LPS) ligation on hADSCs was not modulated by TLR activation (66,67). increased their osteogenic differentiation potential without It appears that TLRs and their ligands can serve as affecting the adipogenic one. In parallel, an increased regulators of MSC immunophenotype and might affect osteogenic differentiation of hADSCs after activation of the maintenance of their immunogenicity state. TLR4 (by its ligand LPS) and TLR2 (by its ligand PGN) is observed in a dose-dependent manner and accompanied by increased ERK activation. Activation of TLR9 (by its MSC multilineage potential and TLR-priming ligand CpG-ODN/2216) inhibited osteogenesis capacity of hADSCs whereas TLR3 activation (by its ligand The multilineage potential of MSCs is considered to Poly(I:C)) and TLR5 (by its ligand flagellin) had no mediate their therapeutic effects during tissue repair effect on it (21). In the case of adipogenic differentiation, underlying their importance in regenerative medicine (68). activation of TLR2 (by its ligand PGN) inhibited it Different studies have reported contrasting effects of TLR significantly, but the other agonists did not affect it. In activation on MSC multilineage potential with promoting addition, Mo et al. found that prolonged LPS challenge versus altering differentiation capacities. for TLR4 activation up-regulated the osteogenic diffe­ TLRs have been shown to be differently involved in rentiation of human BM-MSCs in contrast to TLR2 regulating the differentiation of CB-MSCs (69). Thus, (challenged with lipoteichoic acid) that had no effect adipogenic differentiation was not altered by TLR (73). Furthermore, Pevsner-Fischer et al. (46) showed that activation. TLR2 activation with Pam(3)CSK(4) and TLR signaling may play a distinctive role in regulating TLR4 activation with LPS were able to promote either mouse BM-MSC multipotency depending on the chondrogenesis and osteogenesis of of CB-MSCs but spontaneous versus induced differentiation lineages that with different intensities. In another study, the activation are studied. In non-induced MSC culture, Pam3Cys of TLR3 by Poly(I:C) inhibited the differentiation of activating TLR2 promoted spontaneous osteogenic dif­ UC-MSCs into osteocytes, while that of TLR4 by LPS ferentiation of MSCs and at the same time inhibited increased this differentiation to a certain extent (66). In adipogenic differentiation. In MSC cultures induced to contrast, even expressing high levels of functional TLRs differentiate into the three mesodermal lineages, TLR2 3 and 4, the differentiation potential of BM-MSCs was activation by Pam3Cys significantly reduced mouse BM- not altered after TLR ligation with their respective ligands MSC differentiation into osteoblasts, adipocytes, and (64). However, a recent study demonstrated that TLR4 chondrocytes. Although stimulation with the TLR3 ligand activation by its ligand LPS promoted the osteogenic (Poly(I:C)) promoted the differentiation of mouse BM- differentiation of BM-MSC through Wnt3a and Wnt5a MSCs into the adipocytes and osteoblasts, stimulation signaling (70). Both TLR-3 activated (Poly(I:C)) and with the TLR4 ligand (LPS) induced the reverse effect TLR-4 activated (LPS) were reported to promote osteo­ as it inhibited this process. The transcription factor NF- genesis by favoring the differentiation of BM-MSCs kB, which is triggered by the TLR4-MyD88-dependent to osteoblasts (71). However, these two TLR sub-types pathway was activated and involved in the inhibition of played different roles in different stages of BM-MSC MSCs mesodermal differentiation (74). In a comparative osteogenesis. Zhang and colleagues found that triggering study, TLR ligation was reported to differentially affect

http://immunenetwork.org 93 TLR-priming of MSCs the osteogenic potential of human MSCs depending on requirement for microbiota in the maintenance of steady- their tissue origin (75). Indeed, TLR3 (Poly(I:C)) or TLR4 state hematopoiesis. (LPS) triggering increased the osteogenesis in hADSCs Based on the results in this study, TLR4 (LPS) or NOD1 and, to lesser extent, in BM-MSCs. However, WJ-MSCs ligand-stimulated BM-MSCs are both likely to promote constitutively disclosed a lower osteogenic potential as hematopoiesis. LPS as a potent inducer of IL-6 favors compared with other MSCs, which is not affected by myelopoiesis, whereas NOD1 ligand augments the TLR. numbers of all HSPCs (80). It appears that TLRs and their ligands can serve as It appears that little observations are available for the regulators of MSC differentiation abilities and might impact of TLRs on MSC hematopoiesis support. affect the maintenance of their multipotency state.

MSC immunomodulation and TLR-priming MSC hematopoietic support and TLR-priming Besides not being recognized as immunogenic, MSCs can MSCs play an important role in the physiology and actively sense their surrounding microenvironment and homeostasis of the hematopoietic system (76). MSCs accordingly regulate the function and biology of different support hematopoiesis by creating a niche where HSCs immune cells (81). Thus, MSCs have the capacity to can proliferate and differentiate. Because MSCs gene­ interact with different immune cells from both innate and rate most of the stromal cells present in the niche, and adaptive system and to induce their modulation. produce various molecules regulating hematopoiesis, To achieve their desired effects, MSCs have to be their hematopoiesis-supporting capacity has become recruited to sites of injuries where they will display increasingly important in the treatment of hematologic the functions. Stimulation of TLR present on BM- malignancies during hematopoietic stem cell transplan­ MSC activated downstream signaling pathways with tation (77). Resident mouse BM-MSCs by producing the greatest activation observed for TLR3 (Poly(I:C)). MCP-1 in response to TLR4 activation by LPS was Consequently, and in a TLR-ligand depending manner, reported to induce monocyte emigration from bone this triggering induced the secretion of cytokines and marrow into circulation to confront potential infections chemokines mainly involved in cell migration (48). (78). Brümmendorf and colleagues (79) showed that in Indeed, MSC migration was critically promoted by TLR addition to enhanced myeloid colony formation from ligand exposure with TLR3 as primarily mediating the human CD34 positive cells, TLR4 stimulation by LPS stress migration responses of MSCs when compared to retains overall higher numbers of CD34+ cells in co- TLR2 and TLR9. In line, TLR3 (Poly I : C) and TLR4 culture assays using BM-MSCs, with eightfold more (LPS) triggering have thus converted BM-MSCs into CD34+ cells that underwent up to three divisions as powerfully chemotactic cells capable of enhancing compared to non-stimulated assays. Moreover, CD34+ recruitment of inflammatory immune cells by increasing cells from LPS-stimulated BM-MSC cultures give rise their production of IL-1b, IL-6, IL-8, CCL5 (RANTES), to the full spectrum of myeloid and lymphoid colonies, IP10 and monocyte chemotactic protein (MCP)-1 via thus supporting maintenance of primed hematopoietic activation of NF-kB signaling (51). Similar results have progenitor cells (HPCs) under inflammatory conditions been obtained in AT-MSC, where TLR agonists (PGN for and TLR4 stimulation (79). In BM-MSCs, TLR2 (PAM(3) TLR2 and LPS for TLR4) increased mRNA production CSK(4)) and TLR4 (LPS) activation by their respective of MCP-1 and -2, granulocyte chemotactic protein-2 agonist increased their production of hematopoiesis- (GCP-2), IL-1b, macrophage inflammatory protein-3 a related cytokines promoting thus the proliferation and the (MIP-3 a) (58). Human turbinated MSC (hTMSC) were differentiation of CD34+ cells (52). shown to express relatively high percentage of TLR3 and Recent study by Iwamura et al. (80) shows that re­ TLR4. However, hTMSCs were only immunologically cognition of commensal-derived PAMP by NOD1, but active and responsive to TLR4 as demonstrated by the not NOD2, induced expression of multiple hematopoietic substantial changes in their cytokine and chemokine cytokines (IL-7, FMS-like tyrosine kinase 3 ligand profiles (82). Macrophage-activating ligand-2 (MALP- (Flt3L), stem cell factor (SCF), ThPO, and IL-6) 2), agonist of TLR6 and its known heterodimer partner from BM-MSCs indicating that NOD1 signaling in TLR2, induced activation of NF-kB pathway and lead MSCs serves as an important pathway underlying the AMC to acquire a pro-inflammatory cytokine profile by

94 IMMUNE NETWORK Vol. 17, No. 2: 89-102, April, 2017 TLR-priming of MSCs highly secreting IL-4, IL-6, and IL-8 (56). (Zymosan) and TLR3-agonist (poly I:C) had no effect. Unlike TLR3 (poly I:C), activation of TLR4 with LPS These results suggested that TLR4 and downstream strongly and significantly induced expression of IL- pathways in MSCs exert an important function in B 6, IL-8, IL-12, IP-10 (CXCL10), RANTES (CCL5), lymphocyte-related immune regulation (86). TNF-a, and GM-CSF. In parallel, Tomchuck et al, (83) Once the immune cells are in the surrounding area of demonstrated that upon TLR3 stimulation (Poly(I:C)), MSCs, different regulatory mechanisms can take part in a Janus kinase (JAK) 2/signal transducer and activator the process of immunomodulation. These mechanisms of transcription (STAT) 1 pathway is activated, and the are actively sensitive to the environment of MSCs and expression of suppressor of cytokine signaling (SOCS) thus tightly regulated to allow adequate and efficient proteins is increased. These results further demonstrated response of MSCs. Within the literature, the results about that SOCS1 and SOCS3 play a distinct role in negatively TLR priming and immunomodulation are conflictual and modulating TLR3, JAK/STAT, and CXCR4/CXCR7 reported in very different ways. signaling in BM-MSCs. Collectively, these data suggest Waterman et al. reported a new paradigm for MSC that as negative regulators, SOCS proteins critically immunomodulation functions as they can be specifically affect the way MSC respond to danger signals. These polarized by downstream TLR signaling into two homo­ observations suggest that TLR signaling pathways may genously acting types (65). Indeed, TLR4-primed be manipulated to increase the bio-distribution of infused MSCs (MSC1), mostly producing pro-inflammatory MSCs at the injured sites. mediators (MIP-1a and MIP-1b, RANTES, CXCL9 and Migration and binding of immune cells to MSCs CXCL10), are able to induce T-lymphocyte activation, surrounding environment has been reported to be a key while TLR3-primed MSCs (MSC2), mainly expressing step for establishing immunomodulation (84). Although immunosuppressive factors : IDO (indoleamine-2,3- TLR4 activation with LPS elicited the secretion of pro- dioxygenase), PGE2 (prostaglandin E2), NO (nitric inflammatory mediators CXCL1, IL-6, IL-8, and CCL2, oxide), TGF-b, Hepatocyte Growth Factor (HGF) and Poly (I:C) activating TLR3 increased only the secretion of hemoxygenase (HO), lead to T-cell inhibition. Levin and IL-6 and MIF (macrophage migration inhibitory factor) colleagues (87) investigated the molecular basis for the known to be important in leucocyte recruitment (63). heterogeneity in the response of MSCs to TLR activation. Upon challenge with different TLR ligands on MSCs They found that divergent levels of LPS binding protein isolated from human nasal mucosa (nmMSCs), only (LBP) lead to the heterogenic response of murine BM- activation of TLR3 with its Poly I:C ligand induced the MSCs to TLR activation. In the TLR signaling pathway, strongest release of proinflammatory cytokines (IL-6 and LBP levels predicted the ability of specific MSCs to IL-8) and type I interferon (85). Moreover, under TLR3 secrete pro-inflammatory cytokines in response to LPS. stimulation, mesenchymal stromal cells from human Although some studies reported no significant effect tonsils (T-MSCs) acquire a chemoattractant profile that of TLR activation on AD-MSC, BM-MSC and T-MSC- is suitable for allowing immune cells to migrate into mediated immunosuppression (47,82), others reported MSCs surrounding environment. Indeed, TLR3 activation mitigated observations. Indeed, differently activated increased in the secretion of many chemokine such as MSCs isolated from human nasal mucosa (nmMSCs) CXCL5, CXCL6, CXCL1, CXCL8, and CXCL10 (67). either by TLR3 (poly I:C) or TLR4 (LPS) maintained In terms of leukocyte binding, BM-MSCs responded their ability to suppress leukocyte activation at similar differently to TLR3 and TLR4 activation (63). TLR3 levels, and this effect was shown to be partially mediated pre-activation with Poly I:C significantly increased the by prostaglandins [60]. Liotta et al., demonstrated that number of leukocytes that bind to MSCs, predominantly ligation of TLR3 (poly I:C) and TLR4 (LPS) by their through interacting with hyaluronic acid structures respective ligands impaired the ability of human BM- whereas activation of TLR4 with LPS increased VCAM- MSCs to suppress the proliferation of T-cells (64). These 1 and ICAM-1 dependent binding of leukocytes to MSCs. effects were not associated with alteration of IDO and The expression of B cell activating factor (BAFF), a PGE2 pathways known to be the main mediators of member of the tumor necrosis factor ligand superfamily MSC immunosuppression but rather involved jagged-1 with notable stimulating activity on B cells was inves­ down-regulation induced by TLR3 or TLR4 ligation. tigated in BM-MSCs from both human and murine Indeed, strong evidences indicate that the Jagged-1/Notch species. The BAFF expression was increased in the interaction may be involved in the suppressive activity of presence of TLR4 agonist (LPS), while TLR2 agonist MSCs on T-cell proliferation.

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In contrast, Opitz and colleagues reported that TLR3 these microRNAs were suggested to be involved in the (poly I:C) and TLR4 (LPS) engagement enhanced regulation of BM‑MSC functions. Another study reported the suppressive properties of human BM-MSCs as that, after treating CB-MSCs with various TLR ligands, shown by the increase in their production of regulatory only TLR3 ligand, poly(I:C), significantly improved their kynurenines by the tryptophan-degrading enzyme immunosuppressive abilities by increasing the expression IDO1 (49). Induction of IDO1 by TLR involved an of cyclooxygenase-2 (COX-2) known to be the key autocrine interferon (IFN)-beta signaling loop, which was enzyme in PGE2 production (92). Subsequent results dependent on protein kinase R (PKR), but independent indicated that miR-143 regulates the effect of poly(I:C) of IFN-gamma. In a comparative study, TLR3 (poly I:C) on MSC immunosuppressive function by targeting COX- and TLR4 (LPS) ligation have differentially affected 2 gene. the suppressive functions of BM-, WJ- and AT-MSCs Data from the literature have previously highlighted (54). Remarkably, the immunosuppressive potential of that MSC-mediated T-cell suppression occurs through WJ- and AT-MSC was not affected while BM-MSC the secretion of galectins. In response to TLR2 activa­ showed reduced ability to inhibit lymphocyte activation. tion (PGN), the expression of galectin-3, known to mo­ Differences in the levels of HGF and PGE2 secreted by dulate T-cell biology, was up-regulated at both mRNA MSCs following TLR activation have been hypothesized and protein levels (93) in BM-MSCs. However, such to underline these changes. As shown by Lei J. et al. up-regulation was not linked to a change in MSC immu­ (88), ligation of TLR2 (Pam3Cys) and TLR4 (LPS) on nomodulatory response. Moreover, Gieseke F. et al. (94) BM-MSCs could trigger differential effects on their demonstrated that galectin-9, which is not constitutively immunosuppressive activity. Interestingly, TLR2 but not expressed by BM-MSCs, is strongly induced upon in­ TLR4 activation significantly impaired MSC-mediated teraction with inflammatory cells and functionally immunosuppression of T-cells and reduced MSC- important for the immunosuppressive effects of MSCs. mediated expansion of CD4+CD25+Foxp3+ regulatory Indeed, galectin-9 expression differentially induced by T cells. CXCL10 and iNOS expression and modulation BM-MSCs depending on the TLR ligand activation. by BM-MSCs following TLR activation have proposed Induction of galectin-9 was observed following TLR2 to mediate these effects but should be further clarified. (zymosan), TLR3 (Poly(I:C)) and TLR4 (LPS) activation. Interestingly, it has been suggested that during bacterial In contrast, TLR5 (flagellin) and TLR7/8 (R-848, infection, mouse BM-MSCs may retain a reservoir of the imidazolquinoline compound) did not show any effects on TLR2 ligands (Pam3Cys), in a long-term manner, and galectin-9 expression. Thus, in the presence of a specific release them slowly to maintain an immune response infectious stimuli (TLR activation), BM-MSCs maintain (89). Pam3Cys was transferred from cultured MSCs to their immunosuppressive by inducing the expression of immune cells which induced a pro-inflammatory response galectin-9. In the other hand, the immunomodulatory in vitro and in vivo. In contrast, Rashedi et al. (90) found properties of dental pulp (DP) and dental follicle (DF)- that the generation of Tregs from human lymphocyte MSCs were shown to be differently modulated by TLR cultures was enhanced by either TLR3 (poly I:C) or ligation (57). Activation of TLR3 with Poly(I:C) aug­ TLR4 (LPS) activation of MSCs. This Treg induction was mented the suppressive potential of both cell types by associated with increased gene expression of the Notch potentiating their TGF-b and IL-6 secretion. In contrast, ligand, Delta-like and suggested a new way to enhance TLR4 activation with LPS increased the suppressive the immunomodulatory functions of MSCs. Activation effect of DF-MSCs by enhancing their TGF-b production of TLR4 (LPS) and TLR5 (flagellin) in CB-MSCs do but abrogated that of DP-MSCs by inhibiting their TGF-b not affected their immunosuppressive activity (55). production and IDO-1 expression. These contrasting However, such activation impaired the cytokine secretion effects were suggested to be correlated with the higher profile of unrestricted somatic stem cells (USSCs) expression of TLR3 and TLR4 in DP-MSCs compared independently of TLR ligand nature. Indeed, stimulation with DF-MSCs. Additionally, activation of TLR3 with of USSCs with either LPS or flagellin resulted in a Poly(I:C) enhanced the suppressive functions of T-MSCs marked increase of IL-6 and/or IL-8 production although against Th17 differentiation by increasing programmed levels differed significantly between both stimuli. death ligand-1 (PD-L1) expression (95). Stimulation with TLR2 (PAM3CSK4) or TLR4 (LPS) Moreover, TLR3 preconditioning by Poly(I:C) have been induced a marked expression change in the microRNAs recently demonstrated to enhance the therapeutic efficacy profile of BM‑MSCs (91). The potential target genes of of UC-MSCs via the TLR3-Jagged-1-Notch-1 pathway

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(96). Poly (I:C)-MSCs showed enhanced suppressive 1 ligands by TLR-primed MSC. In addition, TLR3- effects in vitro and in vivo through increasing production primed MSC enhance their suppressive functions against of PGE2 and up-regulation of Jagged-1. Furthermore, NK cells. Furthermore, activation of TLR4 pathway by PGE2 subsequently increased the secretion of IL-10 and LPS ligand has demonstrated more MSC suppressive promoted the differentiation of Treg. In addition, TLR effect towards NK cell proliferation and cytotoxicity activation has been reported to influence the cytokine and thus may provide a potential stroma-targeted tumor balance and thereby controls the outcome of T-cell- therapy (100). Thus, TLR-primed MSCs are able to mediated response (97). As stated Raicevic et al, (53), adapt their immuno-behavior in an inflammatory context TLR activation may affect MSCs immunomodulatory by decreasing their susceptibility to NK killing and by functions by modulating their cytokine profile. Indeed, enhancing their immunosuppressive abilities. a decrease in the immunosuppressive capabilities of It appears that TLRs and their ligands can serve as BM-MSCs is observed following TLR3 and TLR4 acti­ regulators of MSC immunomodulatory capacities but vation by poly(I:C) and LPS respectively. Moreover, the effects are divergent and likely depending on experi­ TLR3 activation augmented IL-6, IL-12p35, IL-23p19, mental settings. and IL-27p28 transcription, whereas TLR4 activation increased IL-23p19 and IL-27p28 transcription. These IL-12 cytokine family members are known to drive Conclusions & perspectives + CD4 T helper 1 (TH1) differentiation and thus promote a T-cell-mediated inflammatory response. TLR3 and Understanding the effects of TLR activation on MSCs TLR4 triggering induced a pro-inflammatory shift in the immunobiology is of great importance to allow efficient cytokine profile of BM-MSCs that should be associated use of their therapeutic effects. In this review, we dis­ with their reduced immunomodulatory functions. cussed and compared the results underlying the rela­ As mentioned in the introduction, MSCs have also tionship between MSCs and activation of different immunomodulatory effects toward cells of the innate TLRs. We focused on the fact that TLRs priming could immune response. Data from Cassatella and colleagues critically influence the phenotype, multilineage potential, revealed that TLR3- and TLR4-activated MSCs diffe­ hematopoietic support and immunomodulation capacity of rently prolonged the survival and function of neutrophils MSCs as they are the main properties of these therapeutic (PMN) (98). Results have showed that TLR3 triggering cells. Despite the large amount of data obtained, there by poly(I:C) dramatically amplifies, in a more significant is great discrepancy of results among the studies. These manner than TLR4 triggering by LPS, the anti-apoptotic differences are probably related to the diversity of effects of activated MSCs in comparison to resting BM- experimental settings that are used to study the impact of MSCs on PMN. In addition, TLR3- and TLR4-activated TLRs on MSCs. In particular, we highlight the important BM-MSCs enhanced the respiratory burst ability and influence of specific culture conditions (e.g. medium, cell CD11b expression by PMN. The biological effects ratio), MSC origin (murine or human), MSC source (e.g. exerted on PMN by TLR3-activated BM-MSCs were bone marrow, cord blood, adipose tissue, etc…), TLR mediated by the combined action of IL-6, IFN-b, and target (e.g. TLR1,2,3, etc…), TLR ligation characteristics GM-CSF, while those exerted by TLR4-activated BM- (e.g. type of ligand, concentration, duration, etc…), and MSCs were mostly depended on GM-CSF. MSCs and the end points to be achieved. Thus, in order to allow safer NK cells have been described to interact in a complex and more efficient therapeutic use of MSCs, the impact manner with bidirectional regulatory effects. Although of infectious conditions and thus TLR activation have to they are able to alter most of activated NK cell biology, be critically studied in well-designed and standardized MSCs are reported to be susceptible to NK-cell mediated assays. Pre-stimulation of MSCs with a specific TLR lysis (17). The results of Giuliani M. et al, showed that ligand may serve as an effective priming step to modulate TLR3-primed (poly(I:C)) BM-MSCs were more resistant one of its function to achieve a desired therapeutic issue. than unprimed MSCs to IL-2-activated NK-induced killing (99). In contrast, no potentialized protection was observed after TLR4 or TLR7/8 priming of BM-MSCs. Conflicts of Interest Such protection can be explained by the modulation of Natural Killer group 2D ligands major histocompatibility The authors declare no potential conflicts of interest. complex class I chain A and ULBP3 and DNAM-

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